JP7539087B2 - Electrode structure and method for producing same - Google Patents

Description

The present invention relates to an electrode structure and a method for manufacturing the same.

In the fields of drug discovery and regenerative medicine, attention has been focused on a technology that uses cells and biomaterials to artificially form three-dimensional cell culture tissues (hereinafter simply referred to as "three-dimensional culture tissues"). For example, three-dimensional culture tissues are expected to be used not only as a drug discovery screening tool to replace conventional animal testing, but also as transplant tissues to replace lost biological tissues.

In three-dimensional cultured tissues, the diffusivity of substances is lower than in two-dimensional cultured tissues of cells (hereinafter simply referred to as "two-dimensional cultured tissues"), so it is difficult to supply nutrients to cells and remove metabolites from cells, and there are concerns that maintaining cell cultures will be difficult. For this reason, there is a demand for tubular cultured vascular tissues to circulate the liquids required for culture inside the tissue, and it has become an important issue to establish a method for efficiently forming three-dimensional cultured vascular tissues that can properly transport substances.

Two important factors in forming three-dimensional cultured vascular tissue are the cylindrical structure and the barrier function of the cultured vascular tissue. The cylindrical structure is a structure necessary as a scaffold when culturing cells. A method of forming a cylindrical structure by culturing cells in a tissue gel, or by culturing cells using a tubular mold, has been investigated (see Non-Patent Document 1). On the other hand, the barrier function is a function that controls the permeation of substances by blood vessels, and is necessary for appropriately transporting substances to the target biological tissue. In cultured vascular tissue, the electrical resistance value of the vascular tissue is widely used as one of the main indicators of the barrier function. For example, a method has been disclosed in which two electrodes are arranged to sandwich vascular tissue (vascular sheet) cultured in a sheet-like shape, and the electrical resistance value of the vascular sheet is measured (see Non-Patent Document 2). In other words, when considering a method of forming a three-dimensional cultured vascular tissue with appropriate functions, a cell culture environment is required in which it is easy to geometrically control the formation of the cylindrical structure of the cultured vascular tissue and to easily measure the electrical resistance value of the vascular sheet.

A. Shimizu et al., Lab Chip, 20 (11), 1917-1927 (2020) B. Srinivasan et al., J. Lab. Autom., 20 (2), 107-126 (2015)

However, with conventional techniques, including those disclosed in Non-Patent Documents 1 and 2, it is difficult to achieve both the formation of a cylindrical structure of cultured vascular tissue and the placement of electrodes, and no prior art capable of resolving this problem has been disclosed. In particular, when forming a cylindrical structure of cultured vascular tissue using tissue gel or a mold, there is no space to place electrodes outside the cylindrical structure, and furthermore, it is difficult to place electrodes inside a cylindrical structure formed on the micrometer (μm) to millimeter (mm) scale.

So far, we have used three-dimensional cultured vascular tissue as an example to explain its formation and evaluation. However, a structure having a cylindrical structure and equipped with electrodes, in which an object for measuring a potential difference by the electrodes can be placed within the cylindrical structure, is not limited to three-dimensional cultured vascular tissue, and can be applied to a variety of objects.

In view of the above circumstances, the present invention aims to provide a structure having a cylindrical structure and equipped with electrodes, in which an object for measuring a potential difference by the electrodes can be placed within the cylindrical structure, and a method for manufacturing the structure.

One aspect of the present invention is an electrode structure comprising a first electrode wound in a cylindrical shape and a second electrode that is not in contact with the first electrode and is wound in a cylindrical or semi-cylindrical shape, the first electrode surrounds the second electrode, the first electrode and the second electrode form a multi-layered cylindrical structure, and the gap between the first electrode and the second electrode in the multi-layered cylindrical structure serves as a cell culture area.

In addition, one aspect of the present invention is a method for manufacturing the electrode structure, which includes a step (A) of forming a first sacrificial layer for laminating the first electrode and a second sacrificial layer for laminating the second electrode on one surface of a substrate that is non-conductive at room temperature, a step (B) of forming the first electrode on the surface of the first sacrificial layer opposite the substrate side, and a step (C) of dissolving the first sacrificial layer and the second sacrificial layer by applying a stimulus from the outside to the first sacrificial layer and liberating the first electrode formed on the first sacrificial layer and the second electrode formed on the second sacrificial layer on the substrate, and rolling the second electrode into a cylindrical or semi-cylindrical shape and rolling the first electrode into a cylindrical shape around the second electrode to form the multilayer cylindrical structure.

The present invention makes it possible to provide a structure having a cylindrical structure and equipped with electrodes, in which an object for measuring a potential difference by the electrodes can be placed within the cylindrical structure, and a method for manufacturing the structure.

FIG. 1 is a perspective view showing a schematic example of an electrode sheet used in the manufacture of an electrode structure according to one embodiment of the present invention. FIG. 2 is a plan view showing a schematic example of a state in which an insulating layer that is an outermost layer, a first support, and a second support are removed from the electrode sheet shown in FIG. 1 . FIG. 1 is a perspective view showing a schematic example of an electrode structure according to an embodiment of the present invention. FIG. 4 is a cross-sectional view showing the electrode structure shown in FIG. 3 in a state in which a circuit is formed. FIG. 2 is a perspective view that illustrates an example of a method for manufacturing an electrode structure according to an embodiment of the present invention. FIG. 2 is a perspective view that illustrates an example of a method for manufacturing an electrode structure according to an embodiment of the present invention. FIG. 2 is a perspective view that illustrates an example of a method for manufacturing an electrode structure according to an embodiment of the present invention. 4 is a graph showing the measurement results of the potential difference between the first electrode and the second electrode in the electrode structure of Example 1. 4 is a graph showing the measurement results of the potential difference between the first electrode and the second electrode in the electrode sheet of Example 1. 1 shows imaging data obtained using a phase-contrast microscope for the three-dimensional cultured vascular tissue prepared in Example 2.

The present invention will be described in detail below with reference to the drawings. Note that the drawings used in the following description may show enlarged essential parts for the sake of convenience in order to make the features of the present invention easier to understand, and the dimensional ratios of each component may not necessarily be the same as in reality.

<<Electrode sheet>>
First, an electrode sheet used in the manufacture of an electrode structure according to an embodiment of the present invention will be described with reference to the drawings. In this specification, unless otherwise specified, the electrode sheet refers to a sheet having an electrode and not having a multilayer cylindrical structure, which is in a stage prior to the construction of the electrode structure according to the present embodiment.

First, the overall configuration of each part in the electrode sheet will be described below.
FIG. 1 is a perspective view illustrating an example of an electrode sheet according to the present embodiment.
The electrode sheet 101 shown here includes a substrate 11, and a first electrode 131 and a second electrode 132 provided on one surface 11a of the substrate 11. The first electrode 131 is in a flat sheet shape, and in the electrode structure described below, it is in a state rolled into a cylindrical shape, and in the electrode sheet 101 shown here, it is in a state before being rolled into a cylindrical shape. The second electrode 132 is in a flat sheet shape, and in the electrode structure described below, it is in a state rolled into a cylindrical or semi-cylindrical shape, and in the electrode sheet 101 shown here, it is in a state before being rolled into a cylindrical or semi-cylindrical shape.

In the electrode sheet 101 , a first sacrificial layer 121 is further provided between the substrate 11 and the first electrode 131 , and a second sacrificial layer 122 is further provided between the substrate 11 and the second electrode 132 .
A first insulating layer 141 is further provided on the surface of the first electrode 131 opposite to the substrate 11 side, and a second insulating layer 142 is further provided on the surface of the second electrode 132 opposite to the substrate 11 side. The first insulating layer 141 and the second insulating layer 142 are both in the form of a flat sheet.
The first sacrificial layer 121 and the second sacrificial layer 122 are layers for temporarily adhering the first electrode 131 and the second electrode 132 to the substrate 11 in the electrode sheet 101 until the time of manufacturing the electrode structure described below. The first sacrificial layer 121 is a layer for laminating the first electrode 131, and the second sacrificial layer 122 is a layer for laminating the second electrode 132. The first sacrificial layer 121 and the second sacrificial layer 122 dissolve when a stimulus is applied from the outside.

The first sacrificial layer 121 , the first electrode 131 and the first insulating layer 141 are all in the form of a film, and in this embodiment, the laminate of the first electrode 131 and the first insulating layer 141 is referred to as a first thin film 91 .
The second sacrificial layer 122 , the second electrode 132 and the second insulating layer 142 are all in the form of a film, and in this embodiment, the laminate of the second electrode 132 and the second insulating layer 142 is referred to as a second thin film 92 .
As described below, the first electrode 131 and the second electrode 132 are arranged at a distance from each other, and in a direction perpendicular to one surface 11a of the substrate 11, a portion where the layered structure of the first electrode 131 and the first insulating layer 141 is not recognized does not correspond to the first thin film 91, and in the same direction, a portion where the layered structure of the second electrode 132 and the second insulating layer 142 is not recognized does not correspond to the second thin film 92, and the boundaries between the first thin film 91 and the second thin film 92 are determined from this perspective.

When viewed from above one surface 11a of the substrate 11 looking down on the first sacrificial layer 121, the first electrode 131, and the first insulating layer 141, the planar shapes of the first sacrificial layer 121, the first electrode 131, and the first insulating layer 141 are all rectangular, and may be either square or rectangular, and the case shown here is rectangular. The planar shapes of the first sacrificial layer 121, the first electrode 131, and the first insulating layer 141 may be other than rectangular, but are preferably rectangular in that the size of the void portion (cell culture portion) in the multilayer cylindrical structure described later becomes larger.
When viewed in a plane as described above, the sizes of the first sacrificial layer 121, the first electrode 131 and the first insulating layer 141 may all be the same, may be only partially the same, or may all be different, but it is preferable that they are all the same in terms of making it easier to manufacture the electrode sheet 101.
When viewed in a plane as described above, the positions of the outer peripheries of the first sacrificial layer 121, the first electrode 131 and the first insulating layer 141 may all be coincident, may only be partially coincident, or may not all be coincident; however, it is preferable that they all be coincident in order to make it easier to manufacture the electrode sheet 101.

The arrangement of the second sacrificial layer 122 , the second electrode 132 and the second insulating layer 142 is similar to the arrangement of the first sacrificial layer 121 , the first electrode 131 and the first insulating layer 141 described above.
That is, when viewed from above one surface 11a side of the substrate 11 looking down on the second sacrificial layer 122, the second electrode 132, and the second insulating layer 142, the planar shapes of the second sacrificial layer 122, the second electrode 132, and the second insulating layer 142 are all rectangular, and may be either square or rectangular, and the case shown here is rectangular. The planar shapes of the second sacrificial layer 122, the second electrode 132, and the second insulating layer 142 may be other than rectangular, but are preferably rectangular in that the size of the void portion (cell culture portion) in the multilayer cylindrical structure described later becomes larger.
When viewed in a plane as described above, the sizes of the second sacrificial layer 122, the second electrode 132 and the second insulating layer 142 may all be the same, may be only partially the same, or may all be different, but it is preferable that they are all the same in terms of making it easier to manufacture the electrode sheet 101.
When viewed in a plane as described above, the positions of the outer peripheries of the second sacrificial layer 122, the second electrode 132 and the second insulating layer 142 may all be coincident, may only be partially coincident, or may not all be coincident; however, in terms of making it easier to manufacture the electrode sheet 101, it is preferable that they all be coincident.

In one region of the one surface 11a of the substrate 11 where the first sacrificial layer 121 and the second sacrificial layer 122 are not provided, a third conductive layer 133 is provided in direct contact with the substrate 11 separately from the first electrode 131 and the second electrode 132, and in the other region, a fourth conductive layer 134 is provided in direct contact with the substrate 11 separately from the first electrode 131 and the second electrode 132. In other words, no sacrificial layer such as the first sacrificial layer 121 or the second sacrificial layer 122 is provided between the substrate 11 and the third conductive layer 133 and between the substrate 11 and the fourth conductive layer 134.
The third conductive layer 133 and the fourth conductive layer 134 are disposed on the substrate 11 and spaced apart from each other.

The first electrode 131 and the third conductive layer 133 are connected by the first axial conductive layer 135. That is, the first electrode 131 and the first axial conductive layer 135 are electrically connected, and the third conductive layer 133 and the first axial conductive layer 135 are electrically connected.
The second electrode 132 and the fourth conductive layer 134 are connected by the second axial conductive layer 136. That is, the second electrode 132 and the second axial conductive layer 136 are electrically connected, and the fourth conductive layer 134 and the second axial conductive layer 136 are electrically connected.

A conductive first contact pad 151 is provided in direct contact with the third conductive layer 133 on a partial region of the surface of the third conductive layer 133 opposite the substrate 11 side. A third insulating layer 143 is provided on a region of the surface of the third conductive layer 133 opposite the substrate 11 side where the first contact pad 151 is not provided. That is, on the third conductive layer 133, the periphery of the first contact pad 151 is surrounded by the third insulating layer 143.
A conductive second contact pad 152 is provided in direct contact with the fourth conductive layer 134 on a partial region of the surface of the fourth conductive layer 134 opposite the substrate 11 side. A fourth insulating layer 144 is provided on a region of the surface of the fourth conductive layer 134 opposite the substrate 11 side where the second contact pad 152 is not provided. In other words, on the fourth conductive layer 134, the second contact pad 152 is surrounded by the fourth conductive layer 134.

When viewed from above one surface 11a of the substrate 11 looking down on the electrode sheet 101, the planar shapes of the third conductive layer 133 and the first contact pad 151 are both rectangular, and may be either a square or a rectangle, and are shown here as being rectangular. The planar shapes of the third conductive layer 133 and the first contact pad 151 may be other than rectangular.

The arrangement of the fourth conductive layer 134 and the second contact pad 152 is similar to the arrangement of the third conductive layer 133 and the first contact pad 151 described above.
That is, when viewed from above the electrode sheet 101 on one surface 11a side of the substrate 11, the planar shapes of the fourth conductive layer 134 and the second contact pad 152 are both rectangular, and may be either a square or a rectangle, and the case shown here is a rectangle. The planar shapes of the fourth conductive layer 134 and the second contact pad 152 may be other than rectangular.

In the electrode sheet 101, the third conductive layer 133 and the first contact pad 151 are electrically connected, so that the first electrode 131, the first axial conductive layer 135, the third conductive layer 133 and the first contact pad 151 are all electrically connected.
Similarly, in the electrode sheet 101, the fourth conductive layer 134 and the second contact pad 152 are electrically connected, and therefore the second electrode 132, the second axial conductive layer 136, the fourth conductive layer 134 and the second contact pad 152 are all electrically connected.

A first axially shaped insulating layer 145 is provided on the surface of the first axially shaped conductive layer 135 opposite the substrate 11 side in direct contact with the first axially shaped conductive layer 135, and in this embodiment, the laminate of the first axially shaped conductive layer 135 and the first axially shaped insulating layer 145 is referred to as the first axial portion 81. The first axial portion 81 has conductivity due to having the first axially shaped conductive layer.
A second axially shaped insulating layer 146 is provided on the surface of the second axially shaped conductive layer 136 opposite the substrate 11 side in direct contact with the second axially shaped conductive layer 136, and in this embodiment, the laminate of the second axially shaped conductive layer 136 and the second axially shaped insulating layer 146 is referred to as the second axial portion 82. The second axial portion 82 has the second axially shaped conductive layer 136 and is therefore conductive.

When viewed from above on one surface 11a of the substrate 11 looking down on the electrode sheet 101, it is preferable that the planar shapes of the first axial conductive layer 135 and the first axial insulating layer 145 are both rectangular (rod-shaped).
When viewed in a plane as described above, the sizes of the first axial conductive layer 135 and the first axial insulating layer 145 may be the same or different, but it is preferable that they are the same in terms of making it easier to manufacture the electrode sheet 101.
When viewed in a plane as described above, the positions of the outer peripheries of the first axial conductive layer 135 and the first axial insulating layer 145 may all be coincident, may only be partially coincident, or may not all be coincident; however, it is preferable that they all be coincident, as this makes it easier to manufacture the electrode sheet 101.
In the first axial portion 81, it is preferable that the first axial conductive layer 135 and the first axial insulating layer 145 are the same size and that the outer periphery positions of the first axial conductive layer 135 and the first axial insulating layer 145 all coincide.

The arrangement of the second axial conductive layer 136 and the second axial insulating layer 146 is similar to the arrangement of the first axial conductive layer 135 and the first axial insulating layer 145 described above.
In other words, when viewed in a plan view looking down on the electrode sheet 101 from above one surface 11a of the substrate 11, it is preferable that the planar shapes of the second axial conductive layer 136 and the second axial insulating layer 146 are both rectangular (rod-shaped).
When viewed in a plane as described above, the sizes of the second axial conductive layer 136 and the second axial insulating layer 146 may be the same or different, but it is preferable that they are the same in terms of making it easier to manufacture the electrode sheet 101.
When viewed in a plane as described above, the positions of the outer peripheries of the second axial conductive layer 136 and the second axial insulating layer 146 may all be coincident, may only be partially coincident, or may not all be coincident; however, it is preferable that they all be coincident, as this makes it easier to manufacture the electrode sheet 101.
In the second axial portion 82, it is preferable that the second axial conductive layer 136 and the second axial insulating layer 146 are the same size and that the outer circumferential positions of the second axial conductive layer 136 and the second axial insulating layer 146 all coincide.

On the one surface 11a of the substrate 11, the stack of the third conductive layer 133, the first contact pad 151, and the third insulating layer 143 is connected to the first thin film 91 by the first shaft portion 81. The stack of the fourth conductive layer 134, the second contact pad 152, and the fourth insulating layer 144 is connected to the second thin film 92 by the second shaft portion 82.

Fig. 2 is a plan view showing an example of the electrode sheet 101 shown in Fig. 1 in a state in which the outermost insulating layers, i.e., the first insulating layer 141, the second insulating layer 142, the third insulating layer 143, the fourth insulating layer 144, the first axial insulating layer 145 and the second axial insulating layer 146, the first support body described later and the second support body described later are removed. The arrangement of each conductive layer in the electrode sheet 101 will be described in more detail with reference to Fig. 2.
In FIG. 2 and subsequent figures, the same components as those shown in the figures already described are given the same reference numerals as in the figures already described, and detailed description thereof will be omitted.

2, the first electrode 131 and the second electrode 132 are disposed apart from each other and are insulated from each other. The outer periphery of the first electrode 131 facing the second electrode 132 and the outer periphery of the second electrode 132 facing the first electrode 131 are parallel to each other.
The first electrode 131 is connected to a first axial conductive layer 135 extending in a direction parallel to one outer periphery of the first electrode 131 (in other words, the longitudinal direction of the first electrode 131). As shown here, a part of the first axial conductive layer 135 on the first electrode 131 side is in contact with the peripheral portion of the first electrode 131 facing the second electrode 132, and at this portion, the first electrode 131 and the first axial conductive layer 135 are integrated. However, no sacrificial layer such as the first sacrificial layer 121 or the second sacrificial layer 122 is provided between the substrate 11 and the first axial conductive layer 135.
The third conductive layer 133 is connected to a first axial conductive layer 135 that is provided extending in a direction parallel to one outer periphery thereof (in other words, the short side direction of the third conductive layer 133).

On the other hand, the second electrode 132 is connected to a second axial conductive layer 136 extending in a direction parallel to one outer periphery of the second electrode 132 (in other words, the longitudinal direction of the second electrode 132). As shown here, a part of the second axial conductive layer 136 on the second electrode 132 side is in contact with the peripheral portion of the second electrode 132 facing the first electrode 131, and at this portion, the second electrode 132 and the second axial conductive layer 136 are integrated. However, no sacrificial layer such as the first sacrificial layer 121 or the second sacrificial layer 122 is provided between the substrate 11 and the second axial conductive layer 136.
The fourth conductive layer 134 is connected to a second axially-shaped conductive layer 136 that is provided extending in a direction parallel to one outer periphery thereof (in other words, the short side direction of the fourth conductive layer 134).
The first axial conductive layer 135 and the second axial conductive layer 136 are preferably disposed apart from each other and parallel to each other.

As shown in FIG. 1, in the electrode sheet 101, a first support 161 and a second support 162 are further provided in a non-contact manner along the longitudinal direction of the first shaft portion 81 on the surface opposite the substrate 11 side of the first shaft portion 81. Both the first support 161 and the second support 162 serve as a base for winding when the first thin film 91 is self-organized and rolled into a cylindrical shape during the manufacture of the electrode structure described below.

The first support 161 is cylindrical, but may be any other shape as long as it is columnar or tubular, for example, prismatic, cylindrical, or rectangular tubular.

It is preferable that the longitudinal direction (in other words, the central axis direction) of the first support 161 and the second support 162 is the same as the longitudinal direction of the first shaft portion 81. It is also preferable that the first support 161 and the second support 162 are arranged in a straight line on the substrate 11.

Of both ends of the first support 161 in the longitudinal direction, the end on the second support 162 side is disposed on a region on the second thin film 92 side of the surface of the first thin film 91 opposite the substrate 11 side. That is, the end of the first support 161 is disposed so as to abut on the first thin film 91. On the other hand, the end of the first support 161 is disposed so as to abut on the second thin film 92.
Of both ends of the second support 162 in the longitudinal direction, the end on the first support 161 side is disposed on a region on the second thin film 92 side of the surface of the first thin film 91 opposite the substrate 11 side. That is, the end of the second support 162 is also disposed so as to abut on the first thin film 91. On the other hand, the end of the second support 162 is not disposed so as to abut on the second thin film 92.
With this arrangement, a gap is formed between the end of the first support 161 and the end of the second support 162. Therefore, in the direction connecting the end of the first support 161 and the end of the second support 162, the entire second thin film 92 is disposed within the gap.

The maximum width of the first support 161 and the second support 162 in a direction perpendicular to their central axis (for example, when the first support 161 and the second support 162 are columnar or cylindrical, the outer diameter (diameter) of the column or cylinder) is preferably 50 to 2000 μm. By having the maximum width in this range, it is possible to more easily create the desired three-dimensional cultured tissue in the multilayer cylindrical structure described below.

As shown in Figures 1 and 2, the first electrode 131 and the first insulating layer 141 are provided with a plurality of first pores 919 that penetrate continuously through them in the stacking direction (in other words, the first thin film 91 penetrates through its thickness direction).

The size of the first pore 919 is not particularly limited as long as it is a size that does not allow a single cell to be cultured to pass through the first pore 919, and can be arbitrarily selected according to the type of cell. In this specification, the "size of the pore" means the size of the opening of the pore in a cross section when a cross section of a thin film (for example, the first thin film 91 in the case of the first pore 919) is formed in a direction perpendicular to the length direction of the pore, or the size of the opening of the pore on the surface of the thin film. The size of the first pore 919 is set in this way in order to prevent the cells to be cultured from leaking out of the multilayer cylindrical structure in the electrode structure described later. For example, when the shape of the opening of the first pore 919 is circular as shown here, the diameter of the opening of the first pore 919 is preferably 6 μm or less. On the other hand, in order to prevent the size of the first pore 919 from being too small, the diameter of the opening of the first pore 919 is preferably 1 μm or more. And, it is more preferable that the diameter of the opening of the first pore 919 is 1 to 6 μm.

Here, the number of first pores 919 is 28, but the number of first pores 919 can be selected arbitrarily according to the purpose, as long as it does not prevent the first thin film 91 from being rolled into a cylindrical shape, as described below. For example, in order to enhance the effect of not preventing the first thin film 91 from being rolled into a cylindrical shape, it is preferable that the ratio of the total area of the openings of the first pores 919 on the surface of the first thin film 91 to the surface area of the first thin film 91 ([total area of the openings of the first pores 919 on the surface of the first thin film 91]/[surface area of the first thin film 91]) is greater than 0 and not greater than 0.5.

As shown in Figures 1 and 2, the second electrode 132 and the second insulating layer 142 have a plurality of second pores 929 that penetrate continuously through them in the stacking direction (in other words, the second thin film 92 penetrates through it in its thickness direction).

The size of the second pore 929 is not particularly limited as long as it is large enough for one cell to be cultured to pass through the second pore 929, and can be selected arbitrarily according to the type of cell. That is, the size of the second pore 929 is larger than the size of the first pore 919. The size of the second pore 929 is set in this way in order to allow the cell to be cultured to move through the second thin film 92 inside the multilayer cylindrical structure in the electrode structure described below. For example, when the shape of the opening of the second pore 929 is circular as shown here, the diameter of the opening of the second pore 929 is preferably 6 μm or more. On the other hand, in order to prevent the size of the second pore 929 from being excessively large, the diameter of the opening of the second pore 929 is preferably 50 μm or less. And, it is more preferable that the diameter of the opening of the second pore 929 is 6 to 50 μm.

Here, the number of second pores 929 is six, but the number of second pores 929 can be selected arbitrarily according to the purpose, as long as it does not prevent the second thin film 92 from being rolled into a cylindrical shape, as described below. For example, in order to enhance the effect of not preventing the second thin film 92 from being rolled into a cylindrical shape, it is preferable that the ratio of the total area of the openings of the second pores 929 on the surface of the second thin film 92 to the surface area of the second thin film 92 ([total area of the openings of the second pores 929 on the surface of the second thin film 92]/[surface area of the second thin film 92]) is greater than 0 and not greater than 0.5.

It is preferable that the outer periphery of the first thin film 91 facing the second thin film 92 and the outer periphery of the second thin film 92 facing the first thin film 91 are parallel to each other.
In a direction perpendicular to the direction in which the first thin film 91 and the second thin film 92 are arranged in parallel (here, the longitudinal direction of the first thin film 91 and the second thin film 92, both of which have rectangular planar shapes), it is preferable that both ends (here, the short sides) of the first thin film 91 protrude more than both ends (here, the short sides) of the second thin film 92.
By using the electrode sheet 101 in which the first thin film 91 and the second thin film 92 are arranged as described above, the electrode structure described below can be manufactured more easily.

As shown in FIG. 1, it is preferable that an insulating layer 14 having the same composition as any of the insulating layers described so far, such as the first insulating layer 141, is filled between the first thin film 91 and the second thin film 92. The electrode sheet 101 provided with the insulating layer 14 in this manner can be manufactured more easily.

The thicknesses of the first electrode 131, the second electrode 132, the third conductive layer 133, the fourth conductive layer 134, the first axial conductive layer 135, and the second axial conductive layer 136 are preferably each independently 0.3 to 10 nm. The thicknesses of the first electrode 131 and the second electrode 132 are preferably equal to or less than the thicknesses of the third conductive layer 133, the fourth conductive layer 134, the first axial conductive layer 135, and the second axial conductive layer 136.
When the thickness of the first electrode 131 is within such a range, by adjusting the ratio of the thickness of the first electrode 131 to the thickness of the first insulating layer 141, it becomes easier to arbitrarily adjust the curvature of the first thin film 91 (in other words, the first electrode described later) in the electrode structure described later when it is wound into a cylindrical shape. The same is true for the second electrode 132. That is, when the thickness of the second electrode 132 is within such a range, by adjusting the ratio of the thickness of the second electrode 132 to the thickness of the second insulating layer 142, it becomes easier to arbitrarily adjust the curvature of the second thin film 92 (in other words, the second electrode described later) in the electrode structure described later when it is wound into a cylindrical shape.
On the other hand, when the thicknesses of the third conductive layer 133, the fourth conductive layer 134, the first axial conductive layer 135 and the second axial conductive layer 136 are within such ranges, the electrode sheet 101 can be manufactured more easily.

The thicknesses of the first insulating layer 141, the second insulating layer 142, the third insulating layer 143, the fourth insulating layer 144, the first axial insulating layer 145, and the second axial insulating layer 146 are preferably each independently 10 to 1000 nm. The thickness of the first insulating layer 141 is preferably thicker than the thickness of the second insulating layer 142, and may be thicker than the thicknesses of the second insulating layer 142, the third insulating layer 143, the fourth insulating layer 144, the first axial insulating layer 145, and the second axial insulating layer 146.
When the thickness of the first insulating layer 141 is within such a range, since the thickness of the first electrode 131 is within the above numerical range, it becomes easier to roll the first thin film 91 (first electrode 131) into a cylindrical shape in a self-organizing manner when manufacturing the electrode structure described later. The same is true for the second insulating layer 142. That is, when the thickness of the second insulating layer 142 is within such a range, since the thickness of the second electrode 132 is within the above numerical range, it becomes easier to roll the second thin film 92 (second electrode 132) into a cylindrical shape in a self-organizing manner when manufacturing the electrode structure described later.
Meanwhile, when the thicknesses of the third insulating layer 143, the fourth insulating layer 144, the first axial insulating layer 145 and the second axial insulating layer 146 are within such ranges, the electrode sheet 101 can be manufactured more easily.

The thickness of the first insulating layer 141 is more preferably 50 to 250 nm. When the thickness of the first insulating layer 141 is in this range, it becomes easier to adjust the curvature of the first thin film 91 (in other words, the first electrode described later) in the electrode structure described later when it is rolled into a cylindrical shape by adjusting the ratio of the thickness of the first electrode 131 to the thickness of the first insulating layer 141. The same applies to the second insulating layer 142. That is, it is more preferably 50 to 250 nm. When the thickness of the second insulating layer 142 is in this range, it becomes easier to adjust the curvature of the second thin film 92 (in other words, the second electrode described later) in the electrode structure described later when it is rolled into a cylindrical shape by adjusting the ratio of the thickness of the second electrode 132 to the thickness of the second insulating layer 142.

The ratio of the thickness of the first electrode 131 to the thickness of the first insulating layer 141 ([thickness of the first electrode 131]/[thickness of the first insulating layer 141]) is preferably 1/3000 to 1/1, and more preferably 1/1200 to 1/4. When the ratio is in such a range, the cylindrically wound state of the first thin film 91 (the first electrode described later) in the electrode structure described later can be more stably maintained.
The ratio of the thickness of the second electrode 132 to the thickness of the second insulating layer 142 ([thickness of the second electrode 132]/[thickness of the second insulating layer 142]) is also preferably 1/3000 to 1/1, and more preferably 1/1200 to 1/4. When the ratio is in such a range, the cylindrically wound state of the second thin film 92 (the second electrode described later) in the electrode structure described later can be more stably maintained.

The first electrode 131, the second electrode 132, the first insulating layer 141, and the second insulating layer 142 are preferably optically transparent. In such a case, cells or three-dimensional cultured tissue in the multilayer cylindrical structure can be more easily observed in the electrode structure described below.

When viewed from above one surface 11a of the substrate 11 looking down on the electrode sheet 101 in a plan view, the size of the first thin film 91 is larger than the size of the second thin film 92. This is because, in the electrode structure described below, the first electrode wound in a cylindrical shape surrounds the second electrode wound in a cylindrical or semi-cylindrical shape, allowing the first electrode and the second electrode to form a multilayer cylindrical structure.

The size of the first thin film 91 and the second thin film 92 can be appropriately selected depending on the type of cells contained in the electrode structure described below, the shape of the desired cultured tissue to be produced by cell culture, etc.

The length of the first thin film 91 in the longitudinal direction (in other words, the length of the outer periphery of the first thin film 91 facing the second thin film 92) is preferably 200 to 10,000 μm.
The length of the first thin film 91 in the short side direction (in other words, the length in the direction perpendicular to the longitudinal direction of the first thin film 91, for example, the length of the periphery of the first thin film 91 facing the third conductive layer 133, or the length of the periphery of the first thin film 91 facing the fourth conductive layer 134) is preferably 20 to 5000 μm. The length of the first thin film 91 in the short side direction is preferably set so that the curvature of the first thin film 91 in its cylindrically wound state, which is determined by the thickness of the first thin film 91, is a desired value.
Here, regardless of whether it is the longitudinal direction or the lateral direction, the length of the first thin film 91 refers to the length of the first thin film 91 when viewed in a planar view looking down on the first thin film 91 from above one surface 11a side of the substrate 11, and if the positions of the outer peripheries of the first electrode 131 and the first insulating layer 141 in the first thin film 91 do not coincide, then one starting point of the length and the other starting point of the length may not be in the same layer.

The longitudinal length of the second thin film 92 (in other words, the length of the outer periphery of the second thin film 92 facing the first thin film 91) is preferably 200 to 10,000 μm, and is preferably shorter than the longitudinal length of the first thin film 91.
The length of the second thin film 92 in the short side direction (in other words, the length in the direction perpendicular to the longitudinal direction of the second thin film 92, for example, the length of the outer periphery of the second thin film 92 on the third conductive layer 133 side or the length of the outer periphery of the second thin film 92 on the fourth conductive layer 134 side among the four outer peripheries) is preferably 20 to 5000 μm, and is preferably shorter than the length of the first thin film 91 in the short side direction. Moreover, it is preferable to set the length of the second thin film 92 in the short side direction so that the curvature of the second thin film 92 in its cylindrically wound state, which is determined by the thickness of the second thin film 92, is a desired value.
Here, the length of the second thin film 92, whether in the longitudinal direction or the transverse direction, is defined in the same manner as for the first thin film 91 described above.

In the first thin film 91, the ratio of the length in the longitudinal direction to the length in the lateral direction ([length in the longitudinal direction of the first thin film 91]/[length in the lateral direction of the first thin film 91]) is preferably 1 to 10. When the ratio is in such a range, the cylindrically wound state of the first thin film 91 (first electrode described later) in the electrode structure described later can be more stably maintained.
In the second thin film 92, the ratio of the length in the longitudinal direction to the length in the lateral direction ([longitudinal length of the second thin film 92]/[length in the lateral direction of the second thin film 92]) is preferably 1 to 10. When the ratio is in such a range, the cylindrically wound state of the second thin film 92 (second electrode described later) in the electrode structure described later can be more stably maintained.

The lengths in the longitudinal and lateral directions of the first sacrificial layer 121, the first electrode 131, and the first insulating layer 141 may be, for example, the same as the lengths in the longitudinal and lateral directions of the above-mentioned first thin film 91.
The lengths in the longitudinal and lateral directions of the second sacrificial layer 122, the second electrode 132, and the second insulating layer 142 may be, for example, the same as the lengths in the longitudinal and lateral directions of the second thin film 92 described above.

The distance between the first electrode 131 and the second electrode 132 is preferably 10 μm or more in terms of stably maintaining insulation between the first electrode 131 and the second electrode 132. On the other hand, the distance between the first electrode 131 and the second electrode 132 is preferably 1000 μm or less in terms of avoiding this distance becoming excessive. And, the distance between the first electrode 131 and the second electrode 132 is more preferably 10 to 1000 μm.
In this specification, the distance between a first electrode and a second electrode means the minimum distance between a point on the outer circumference of the first electrode and a point on the outer circumference of the second electrode, and in the electrode sheet 101, it is the minimum distance between a point on the outer circumference of the first electrode 131 facing the second electrode 132 and a point on the outer circumference of the second electrode 132 facing the first electrode 131.
In addition, when the first axial conductive layer 135 or the second axial conductive layer 136 is present between the first electrode 131 and the second electrode 132, it is preferable that the distance between the first electrode 131 and the second axial conductive layer 136 or the distance between the second electrode 132 and the first axial conductive layer 135 is similar to the above-mentioned distance between the first electrode 131 and the second electrode 132. The distance between the first electrode 131 and the second axial conductive layer 136 and the distance between the second electrode 132 and the first axial conductive layer 135 are also defined in the same manner as the above-mentioned distance between the first electrode and the second electrode.

Even when the second axial conductive layer 136 is not present between the first electrode 131 and the second electrode 132, it is preferable that the distance between the first electrode 131 and the second axial conductive layer 136 is the same as the distance between the first electrode 131 and the second electrode 132 described above. The reason for this is the same as in the case of the distance between the first electrode 131 and the second electrode 132 described above.

The widths of the first shaft portion 81 and the second shaft portion 82 are not particularly limited as long as the first thin film 91 (first electrode 131) and the second thin film 92 (second electrode 132) are not prevented from being rolled into a cylindrical shape in a self-organizing manner during the manufacture of the electrode structure described later. For example, the widths of the first shaft portion 81 and the second shaft portion 82 are preferably 50 μm or more, respectively. By having the widths of the first shaft portion 81 and the second shaft portion 82 in such a range, the breakage of these shaft portions due to the stress generated during the deformation of the first thin film 91 and the second thin film 92 is highly suppressed. On the other hand, the widths of the first shaft portion 81 and the second shaft portion 82 are preferably 200 μm or less, respectively. By having the widths of the first shaft portion 81 and the second shaft portion 82 in such a range, the electrode sheet 101 and the electrode structure described later can be made smaller. And, it is more preferable that the widths of the first shaft portion 81 and the second shaft portion 82 are each independently 50 to 200 μm.
Here, the width of the first shaft portion 81 refers to the width of the first shaft portion 81 when viewed in a plan view looking down on the first shaft portion 81 from above one surface 11a of the substrate 11, and if the positions of the outer circumferences of the first axial conductive layer 135 and the first axial insulating layer 145 in the first shaft portion 81 do not coincide, the starting point of one side of the width and the starting point of the other side may not be in the same layer. The width of the second shaft portion 82 is defined in the same way as for the first thin film 91.

The longitudinal and lateral lengths of the first contact pad 151 are preferably each independently 1000 to 5000 μm. With the longitudinal and lateral lengths in such ranges, it is easier to bring a probe into contact with the first contact pad 151 when measuring the potential difference between the first electrode and the second electrode in the electrode structure described below. However, if the planar shape of the first contact pad 151 is rectangular, the longitudinal length is longer than the lateral length.

The thickness of the first contact pad 151 is not particularly limited, but is preferably 100 nm or more. With the thickness of the first contact pad 151 in this range, even if the operation of contacting the first contact pad 151 with a probe is repeated when measuring the potential difference between the first electrode and the second electrode in the electrode structure described later, damage to the first contact pad 151 is highly suppressed. On the other hand, the thickness of the first contact pad 151 is preferably 2000 μm or less. With the thickness of the first contact pad 151 in this range, the electrode sheet 101 and the electrode structure described later can be made thinner. And, the thickness of the first contact pad 151 is more preferably 100 nm to 2000 μm.

The thickness of the second contact pad 152 is preferably the same as that of the first contact pad 151 for the same reasons as those for the first contact pad 151. That is, the thickness of the second contact pad 152 is preferably 100 nm or more, preferably 2000 μm or less, and more preferably 100 nm to 2000 μm.

The lengths of the third conductive layer 133 and the fourth conductive layer 134 in the longitudinal and lateral directions are preferably, for example, independently 1000 to 7000 μm. However, when the planar shape of the third conductive layer 133 or the fourth conductive layer 134 is rectangular, the length in the longitudinal direction is longer than the length in the lateral direction.

The thickness of the first sacrificial layer 121 and the second sacrificial layer 122 is not particularly limited. For example, in order to dissolve the first sacrificial layer 121 or the second sacrificial layer 122 more quickly when a stimulus is applied to the first sacrificial layer 121 or the second sacrificial layer 122, it is preferable that the thickness of the first sacrificial layer 121 and the second sacrificial layer 122 is each independently 20 to 1000 nm.

In the electrode sheet 101 , the substrate 11 carries a first thin film 91 , a second thin film 92 , a first shaft portion 81 and a second shaft portion 82 .
The size of the substrate 11 is not particularly limited as long as it is capable of supporting these.
The thickness of the substrate 11 is not particularly limited, but is preferably 50 to 200 μm.

Next, the constituent materials of each part of the electrode sheet will be described below.
The first electrode 131, the second electrode 132, the third conductive layer 133, the fourth conductive layer 134, the first axial conductive layer 135, and the second axial conductive layer 136 have sufficient conductivity. As long as these conditions are met, the constituent materials of these conductive layers (in other words, conductive materials) are not particularly limited.
The constituent materials of these conductive layers, particularly the constituent materials of the first electrode 131 and the second electrode 132, are preferably capable of forming a thin film, preferably have no or only a small change in volume when immersed in a solution, preferably have high optical transparency, and preferably have no or low cellular activity.

Specific examples of the constituent materials (conductive materials) of the first electrode 131, the second electrode 132, the third conductive layer 133, the fourth conductive layer 134, the first axial conductive layer 135, and the second axial conductive layer 136 include conductive carbon materials such as graphene and carbon nanotubes; planar materials such as molybdenum disulfide, etc.

The constituent materials (conductive materials) of the first electrode 131, the second electrode 132, the third conductive layer 133, the fourth conductive layer 134, the first axial conductive layer 135, and the second axial conductive layer 136 may each be only one type, or may be two or more types. If there are two or more types, the combination and ratio of the materials can be selected arbitrarily according to the purpose. However, in this embodiment, it is preferable that the constituent materials of these conductive layers are only one type.

The constituent materials (conductive materials) of the first electrode 131, the second electrode 132, the third conductive layer 133, the fourth conductive layer 134, the first axial conductive layer 135, and the second axial conductive layer 136, particularly the constituent materials of the first electrode 131 and the second electrode 132, are preferably conductive carbon materials, and more preferably graphene. Graphene is highly biocompatible and highly transparent, so that in the electrode structure described below, cells or three-dimensional cultured tissues in the multilayer cylindrical structure can be more easily observed by a fluorescent microscope. The light transmittance of graphene is 97.7%, and graphene has a higher light transmittance than conductive metal materials such as gold, silver, and copper.

The first insulating layer 141, the second insulating layer 142, the third insulating layer 143, the fourth insulating layer 144, the first axial insulating layer 145, and the second axial insulating layer 146 are either non-conductive or have extremely low conductivity to the extent that they can be regarded as non-conductive. As long as these conditions are met, the constituent materials of these insulating layers (in other words, insulating materials) are not particularly limited.
The constituent materials of these insulating layers, particularly the first insulating layer 141 and the second insulating layer 142, are preferably capable of forming a thin film, preferably have no or only a small change in volume when immersed in a solution, preferably have high optical transparency, and preferably have no or low cellular activity.

More specifically, examples of the constituent materials (insulating materials) of the first insulating layer 141, the second insulating layer 142, the third insulating layer 143, the fourth insulating layer 144, the first axial insulating layer 145 and the second axial insulating layer 146 include polymeric compounds such as polyparaxylylene, polyparaxylylene derivatives, polyimide, and various photoresists; inorganic oxides such as silicon dioxide (SiO ₂ ) and aluminum oxide (also known as alumina, Al ₂ O ₃ ); and the like.
Examples of the polyparaxylene derivative include polyhalogenated paraxylenes such as polychloroparaxylene and polyfluoroparaxylene.

The constituent materials (insulating materials) of the first insulating layer 141, the second insulating layer 142, the third insulating layer 143, the fourth insulating layer 144, the first axial insulating layer 145, and the second axial insulating layer 146 may each be only one type, or may be two or more types. If there are two or more types, the combination and ratio of the materials can be selected arbitrarily according to the purpose. However, in this embodiment, it is preferable that the constituent materials of these conductive layers are only one type.

For example, when the conductive layer is made of graphene (conductive material) and the insulating layer is made of polyparaxylylene or a polyparaxylylene derivative, many aromatic rings are present in the molecules of polyparaxylylene or a polyparaxylylene derivative, and an interaction between π electrons occurs between these aromatic rings and graphene, resulting in high adhesion between the conductive layer and the insulating layer. Therefore, for example, when the first thin film 91 or the second thin film 92 is rolled into a cylindrical shape in a self-organizing manner as described below, peeling between the first electrode 131 and the first insulating layer 141, and peeling between the second electrode 132 and the second insulating layer 142 can be highly suppressed.

In this specification, when a structure in which one or more hydrogen atoms in a certain specific compound are replaced with a group other than a hydrogen atom is assumed, the compound having such a replaced structure is referred to as a "derivative" of the above-mentioned specific compound.
In this specification, unless otherwise specified, the term "group" includes not only an atomic group having a structure in which a plurality of atoms are bonded, but also a single atom.

A protein layer may be provided on the surface (the surface opposite to the first electrode 131) of the first insulating layer 141 in the first thin film 91. The provision of the protein layer on the surface of the first insulating layer 141 allows cells to adhere thereto, thereby improving the adhesion of cells.
The protein layer is a layer containing a protein as a main component, and for example, in the protein layer, the ratio of the protein content to the total mass of the protein layer is preferably 50% by mass or more, while the ratio is 100% by mass or less.
Examples of the protein include proteins that function as extracellular matrices, such as fibronectin, collagen, and laminin.
The protein layer may contain only one type of protein, or two or more types of proteins. When the protein layer contains two or more types of proteins, the combination and ratio of the proteins can be arbitrarily selected depending on the purpose.

As in the case of the first thin film 91, a protein layer may or may not be provided on the surface of the second insulating layer 142 in the second thin film 92 (the surface opposite the second electrode 132 side), but it is usually sufficient that a protein layer is provided only on the first thin film 91.

The constituent materials of the first sacrificial layer 121 and the second sacrificial layer 122 are not particularly limited as long as they have the property of dissolving when an external stimulus is applied thereto. Examples of the stimulus include the action of a chemical substance, a temperature change, and light irradiation.
More specifically, the first sacrificial layer 121 and the second sacrificial layer 122 may be made of, for example, calcium alginate gel, which is a type of physical gel. Calcium alginate gel is transformed into a sol and dissolved by the addition of a chelating agent such as sodium citrate or ethylenediaminetetraacetic acid (EDTA), or an enzyme such as alginate lyase. Alginate lyase is particularly preferred because it has little effect on cell survival.

The first support 161 and the second support 162 have insulating properties. As long as these conditions are met, the materials constituting these supports (in other words, insulating materials) are not particularly limited.
More specifically, examples of the constituent materials of the first support 161 and the second support 162 include borosilicate glass, silicone rubber, and epoxy resin.
The outer surface of the first support 161 (in other words, the surface in contact with the first thin film 91 in the electrode structure described later) may be coated with a hydrophobic material, a charged polymer, or the like. For example, when the material of the first insulating layer 141 in the first thin film 91 is polyparaxylylene or a polyparaxylylene derivative, the surface of the first insulating layer 141 (the surface opposite to the first electrode 131) is negatively charged, and therefore, when the outer surface of the first support 161 is coated with a cationic polymer, the adhesion between the first support 161 and the first insulating layer 141 is improved in the electrode structure described later. Examples of the cationic polymer include polyethyleneimine, polylysine, polyornithine, and the like, which are also suitable in that they do not affect cell activity.

The first contact pad 151 and the second contact pad 152 have sufficient conductivity. As long as this condition is satisfied, the material (in other words, the conductive material) that constitutes these contact pads is not particularly limited.
More specifically, examples of materials that can be used to form the first contact pad 151 and the second contact pad 152 include gold, copper, platinum, aluminum, and chromium.

The material constituting the substrate 11 is not particularly limited as long as it is non-conductive at room temperature, but it is preferable that the material be one that can increase the flatness of the substrate 11, and that in the electrode structure described below, one that does not interfere with the observation of cells or three-dimensional cultured tissue in the multilayer cylindrical structure by a fluorescent microscope.
More specifically, examples of the constituent material of the substrate 11 include glass such as borosilicate glass, resin such as polystyrene, silicon, and the like.
In this specification, "room temperature" means a temperature that is neither particularly cooled nor heated, that is, an ordinary temperature, and examples thereof include temperatures of 15 to 25°C.

<<Electrode structure>>
Next, an electrode structure according to an embodiment of the present invention will be described with reference to the drawings. The electrode structure according to the present embodiment is obtained by using the electrode sheet described above.
3 is a perspective view showing an example of the electrode structure of the present embodiment. The electrode structure 1 shown here can be manufactured using the electrode sheet 101 shown in FIG.

The electrode structure 1 includes a first electrode 1310 wound in a cylindrical shape, and a second electrode 1320 that is provided in a non-contact state with the first electrode 1310 and that is wound in a cylindrical or semi-cylindrical shape.
The first electrode 1310 is the same as the first electrode 131 described above, except that it is wound in this manner, and the second electrode 1320 is the same as the second electrode 132 described above, except that it is wound in this manner.
In the electrode structure 1, the first electrode 1310 surrounds the second electrode 1320, and the entire semi-cylindrical second electrode 1320 is contained in the hollow portion of the cylindrical first electrode 1310. As a result, the first electrode 1310 and the second electrode 1320 form a two-layer cylindrical structure 90.

Here, the second electrode 1320 is wound in a semi-cylindrical shape, but it may be wound in a cylindrical shape like the first electrode 1310.

More specifically, the first electrode 1310 is wound into a cylindrical shape in the state of the first thin film 910, which is a laminate with the first insulating layer 1410. That is, in the first electrode 1310, the exposed surface of the first electrode 1310 faces the outside of the cylinder (is a convex surface), and the surface of the first electrode 1310 facing the first insulating layer 1410 faces the inside of the cylinder (is a concave surface). The first insulating layer 1410 is the same as the first insulating layer 141 described above, except that it is wound in this way, and the first thin film 910 is the same as the first thin film 91 described above, except that it is wound in this way. That is, the first thin film 910 is configured by laminating the first insulating layer 1410 on the radially inward surface of the two-layer cylindrical structure 90 of the first electrode 1310.

More specifically, the second electrode 1320 is wound in a semi-cylindrical shape in the state of the second thin film 920, which is a laminate with the second insulating layer 1420. That is, in the second electrode 1320, the exposed surface of the second electrode 1320 faces the outside of the semi-cylinder (it is a convex surface), and the surface of the second electrode 1320 facing the second insulating layer 1420 faces the inside of the semi-cylinder (it is a concave surface). The second insulating layer 1420 is the same as the second insulating layer 142 described above, except that it is wound in this way, and the second thin film 920 is the same as the second thin film 92 described above, except that it is wound in this way. That is, the second thin film 920 is configured by laminating the second insulating layer 1420 on the surface of the second electrode 1320 facing inward in the radial direction of the two-layer cylindrical structure 90.

The gap between the first electrode 1310 and the second electrode 1320 in the two-layer cylindrical structure 90 (in other words, the gap between the first thin film 910 and the second thin film 920) has a suitable size and can be filled with culture medium. In addition, the first insulating layer 1410 that serves as a scaffold for the cells 7 to attach is also present, making it suitable as a cell culture section.

In the electrode structure 1, the first thin film 910 is connected to the first shaft portion 81, the first shaft portion 81 is fixed to the substrate 11, and the first thin film 910 is wound around the first shaft portion 81 as a fulcrum. On the other hand, the second thin film 920 is connected to the second shaft portion 82, the second shaft portion 82 is fixed to the substrate 11, and the second thin film 920 is wound around the second shaft portion 82 as a fulcrum.

In this way, in the electrode structure 1, the first thin film 910, which is wound in a cylindrical shape, surrounds the second thin film 920, which is wound in a semi-cylindrical shape, and the first thin film 910 and the second thin film 920 can be said to form a two-layer cylindrical structure 90.

In this specification, the term "a first electrode wound in a cylindrical shape" means that the shape of the first electrode is completely cylindrical, or that the shape is wound to a degree that can be regarded as cylindrical at a glance, although it cannot be said to be completely cylindrical. When the shape of the first electrode is completely cylindrical, no gap is observed between the outer circumferences of the first electrodes, and when the shape of the first electrode is not completely cylindrical, a gap is observed between the outer circumferences of the first electrodes, but when this gap is extremely small, the first electrodes are regarded as being wound in a cylindrical shape. These points are also true for the "second electrode wound in a cylindrical shape", "first thin film wound in a cylindrical shape", and "second thin film wound in a cylindrical shape".
The term "second electrode wound in a semi-cylindrical shape" means that the second electrode has a shape that is completely semi-cylindrical (a shape obtained by dividing a cylinder in two by a plane including its central axis), or that is wound to a degree that can be regarded as semi-cylindrical at a glance, although it cannot be said to be completely semi-cylindrical. The same applies to the "second thin film wound in a semi-cylindrical shape".

The ratio of the circumferential length of the cylindrical first thin film 910 in the direction in which it is wound to the lateral length of the first thin film 910 in a flat state (i.e., the first thin film 91 before being rolled into a cylindrical shape) ([circumferential length of the cylindrical first thin film 910 in the direction in which it is rolled]/[lateral length of the first thin film 91 before being rolled into a cylindrical shape]) is preferably 0.7 to 1.5. By having the ratio in this range, the shape of the cylindrically rolled first thin film 910 can be more stably maintained. If the shape of the first thin film 910 is not completely cylindrical, the circumferential length is determined by eliminating any gaps that exist between the peripheries of the first thin film 910.

The ratio of the circumferential length of the cylindrical or semi-cylindrical second thin film 920 in the direction of its roll to the lateral length of the second thin film 920 in a flat state (i.e., the second thin film 92 before being rolled into a cylindrical or semi-cylindrical shape) ([circumferential length of the cylindrical or semi-cylindrical second thin film 920 in the direction of its roll]/[lateral length of the second thin film 92 before being rolled into a cylindrical or semi-cylindrical shape]) is preferably 0.7 to 1.5. By setting the ratio in this range, the shape of the second thin film 920 rolled into a cylindrical or semi-cylindrical shape can be more stably maintained. The circumferential length of the cylindrical or semi-cylindrical second thin film 920 is specified in the same manner as the circumferential length of the cylindrical first thin film 910.

The maximum width of the cylindrical second thin film 920 in a direction perpendicular to its central axis (e.g., if the second thin film 920 is cylindrical, the outer diameter (diameter) of the cylinder) is not particularly limited as long as it is smaller than the maximum width of the cylindrical first thin film 910 in a direction perpendicular to its central axis (e.g., if the first thin film 910 is cylindrical, the outer diameter (diameter) of the cylinder). If the second thin film 920 is semi-cylindrical, it is preferable that the cylindrical second thin film 920 is the same size as the cylindrical second thin film 920 described above, assuming that the second thin film 920 becomes cylindrical with the same curvature.

The maximum width of the cylindrical first thin film 910 in the direction perpendicular to its central axis (for example, if the first thin film 910 is cylindrical, the outer diameter of the cylinder) is preferably 50 to 2000 μm. By keeping the maximum width in this range, the desired three-dimensional cultured tissue can be more easily produced in the two-layer cylindrical structure 90.

The ratio of the maximum width of the cylindrical first thin film 910 to the maximum width of the first support 161 ([maximum width of the cylindrical first thin film 910 in a direction perpendicular to its central axis]/[maximum width of the first support 161 in a direction perpendicular to its central axis]) is preferably 1 to 1.1. The first thin film 910 having such a ratio in this range is wound with high precision so that its shape and size are as intended or very close to the intended shape and size.
The ratio of the maximum width of the cylindrical first thin film 910 to the maximum width of the second support 162 is also similar.

The ratio of the curvature of the second thin film 920 to the curvature of the first thin film 910 ([curvature of the second thin film 920]/[curvature of the first thin film 910]) is preferably 0 to 0.8. With the ratio in this range, the desired three-dimensional cultured tissue can be more easily produced in the two-layer cylindrical structure 90.

The ratio of the length of the cylindrical or semi-cylindrical second thin film 920 in its central axis direction to the length of the cylindrical first thin film 910 in its central axis direction ([length of the cylindrical or semi-cylindrical second thin film 920 in its central axis direction]/[length of the cylindrical first thin film 910 in its central axis direction]) is preferably greater than 0 and less than or equal to 0.8. By having the ratio in this range, the desired three-dimensional cultured tissue can be more easily produced in the two-layer cylindrical structure 90.

The positional relationship between the first electrode 131 and the first shaft portion 81 in the electrode sheet 101 is maintained as is in the electrode structure 1. Therefore, in the electrode structure 1, it is preferable that the first electrode 1310 is connected to the first shaft portion 81 that is provided extending in a direction perpendicular to the winding direction (in other words, in the central axis direction of the cylindrical first electrode 1310).
The same is true for the second electrode 1320, and in the electrode structure 1, it is preferable that the second electrode 1320 is connected to a second axis portion 82 extending in a direction perpendicular to the winding direction (in other words, in the central axis direction of the cylindrical second electrode 1320).

As explained above, the first shaft portion 81 has the first axial conductive layer 135, and the second shaft portion 82 has the second axial conductive layer 136, so that the first electrode 1310 and the first axial conductive layer 135 are electrically connected, and the second electrode 1320 and the second axial conductive layer 136 are electrically connected. Therefore, in the electrode structure 1, the potential difference between the first electrode 1310 and the second electrode 1320 can be measured via the first shaft portion 81 and the second shaft portion 82.
Furthermore, in the electrode structure 1, the first electrode 1310, the first axial conductive layer 135, the third conductive layer 133, and the first contact pad 151 are all electrically connected. The second electrode 1320, the second axial conductive layer 136, the fourth conductive layer 134, and the second contact pad 152 are also all electrically connected. Therefore, by contacting a probe with the first contact pad 151 and the second contact pad 152, respectively, the electrode structure 1 can be connected to various voltmeters or ammeters, and for example, the potential difference between the first electrode 1310 and the second electrode 1320 can be measured.

Figure 4 is a cross-sectional view that shows a schematic cross section of the electrode structure 1. Here, an ammeter is connected to the electrode structure 1 to form a circuit. However, some components of the electrode structure 1 are not shown in Figure 4.

In the electrode structure 1, each component is the same as in the case of the electrode sheet 101 described above, except for the points described so far. Therefore, further detailed explanations of each component are omitted.

When cells are cultured in the gap between the first thin film 910 and the second thin film 920 in the two-layer cylindrical structure 90, the cells grow along the surface of the first thin film 910 on the second thin film 920 side, forming a three-dimensional cultured tissue. This three-dimensional cultured tissue has a cylindrical structure that reflects the structure of the two-layer cylindrical structure 90. A first electrode 1310 is present in the first thin film 910, and a second electrode 1320 is present in the second thin film 920. Therefore, in the electrode structure 1, the electrical resistance value of the three-dimensional cultured tissue can be measured by measuring the potential difference between the first electrode 1310 and the second electrode 1320.

If the first support 161 and the second support 162 are cylindrical rather than columnar, the hollow portion can be filled with culture fluid during cell culture, and the culture state can be maintained in a healthier state by perfusing the culture fluid. However, the culture fluid in the hollow portion is adjusted so that it does not mix with the culture fluid outside the two-layer cylindrical structure 90.

The cells to be cultured in the electrode structure 1 are not particularly limited and can be selected arbitrarily depending on the purpose. More specifically, examples of the cells include human umbilical vein endothelial cells (HUVECs) and brain microvascular endothelial cells.

When three-dimensional cultured vascular tissue is produced using the electrode structure 1, for example, the so-called barrier function that controls the permeation of substances through blood vessels can be evaluated by measuring the electrical resistance value. After producing an appropriate three-dimensional cultured vascular tissue, when culturing other cells, this three-dimensional cultured vascular tissue can be used to appropriately transport nutrients and other substances to the other cells, and also to appropriately transport waste products such as metabolites of other cells.

For example, about five days after the start of culture, HUVECs form bonds between cells and develop a barrier function. Therefore, by measuring the electrical resistance on the first day of culture and several days after culture has started, and comparing the difference, the barrier function of the HUVECs that have formed a cylindrical structure can be evaluated. When glass tubes or the like are used as the first support 161 and the second support 162, it is also possible to deliver a fluorescent indicator to evaluate the permeability of a drug to the three-dimensional cultured vascular tissue. However, the above is merely one example of the use of the electrode structure 1.

In the electrode structure 1, a first electrode 1310 and a second electrode 1320 are arranged inside a two-layer cylindrical structure 90 formed on the micrometer (μm) to millimeter (mm) scale, and further, three-dimensional cultured vascular tissue can be arranged, making it possible to realize a structure never before seen.

Here, the formation and evaluation of three-dimensional cultured vascular tissue has been explained as an example, but the object for measuring the potential difference that can be placed inside the two-layered cylindrical structure 90 is not limited to three-dimensional cultured vascular tissue, and the present invention can be applied to a variety of objects.

So far, the electrode structure of this embodiment has been described as having a two-layer cylindrical structure, but it may have a multi-layer cylindrical structure of three or more layers instead of two layers. In an electrode structure having a multi-layer cylindrical structure of three or more layers, the number of electrodes (first electrode 1310 and second electrode 1320 in the electrode structure 1) per unit volume can be increased, making it possible to measure a higher level of potential difference.
In this specification, all cylindrical structures having two or more layers are referred to as "multi-layer cylindrical structure."

An example of an electrode structure having a multilayer cylindrical structure of three or more layers is one in which a second thin film is wound in a spiral shape in two or more layers, surrounded by a cylindrical first thin film as described above.
An electrode structure having a multi-layered cylindrical structure of three or more layers may be similar to the electrode structure having a two-layered cylindrical structure described above, except that the number of layers of the cylindrical structure is different.

As described above, the electrode structure of this embodiment has a multilayer cylindrical structure of two or more layers and is equipped with a first electrode and a second electrode, and objects such as cells for measuring the potential difference and electrical resistance value using the first electrode and the second electrode can be placed (contained) within the multilayer cylindrical structure.

The electrode structure of this embodiment is suitable for use as a biological electrode for measuring the potential difference and electrical resistance value of not only three-dimensional cultured vascular tissue but also biomaterials such as cells and biological tissue.
Furthermore, by disposing (containing) something other than the biomaterial within the multi-layered cylindrical structure, the electrode structure of this embodiment can be used for purposes other than as a bioelectrode.

<<Method of Manufacturing Electrode Structure>>
A manufacturing method of the electrode structure of this embodiment includes a step (A) of forming the first sacrificial layer (a first sacrificial layer for laminating the first electrode) and the second sacrificial layer (a second sacrificial layer for laminating the second electrode) on one surface of the substrate (a substrate that is non-conductive at room temperature), which is non-conductive at room temperature; a step (B) of forming the first electrode on the surface of the first sacrificial layer opposite the substrate side, and forming the second electrode on the surface of the second sacrificial layer opposite the substrate side; and a step (C) of dissolving the first sacrificial layer and the second sacrificial layer by applying a stimulus from the outside to the first sacrificial layer and the second sacrificial layer, liberating the first electrode formed on the first sacrificial layer and the second electrode formed on the second sacrificial layer on the substrate, and winding the second electrode into a cylindrical or semi-cylindrical shape and winding the first electrode into a cylindrical shape around the second electrode to form the multilayer cylindrical structure.
The electrode structure of this embodiment can be manufactured, for example, by producing the electrode sheet, and then winding the second electrode into a cylindrical or semi-cylindrical shape on the electrode sheet, and winding the first electrode into a cylindrical shape surrounding the second electrode, thereby forming the multilayer cylindrical structure.
Hereinafter, a method for manufacturing the electrode structure of this embodiment will be described with reference to the drawings.

Figures 5 and 6 are perspective views that show a schematic example of a method for manufacturing an electrode structure according to this embodiment. Here, the manufacturing method will be explained using the electrode structure 1 shown in Figure 3 as an example.

<Step (A)>
In the step (A), a first sacrificial layer 121 and a second sacrificial layer 122 are formed on one surface 11a of a substrate 11, as shown in FIG. 5(a).
There is no particular limitation on the method of forming the first sacrificial layer 121 and the second sacrificial layer 122, and the method can be arbitrarily selected depending on the type of constituent material of the first sacrificial layer 121 and the second sacrificial layer 122. Examples of the method of forming the first sacrificial layer 121 and the second sacrificial layer 122 include a typical thin film forming method, and more specifically, for example, a chemical vapor deposition (CVD) method, a spin coating method, an inkjet printing method, a vapor deposition method, an electrospray method, etc.

In step (A), for example, a sacrificial layer may be formed only in the area on one surface 11a of the substrate 11 where the first electrode 131 and the second electrode 132 are to be formed, and these sacrificial layers may be the first sacrificial layer 121 and the second sacrificial layer 122. In addition, after forming a sacrificial layer in the area on one surface 11a of the substrate 11 where the first electrode 131 and the second electrode 132 are to be formed and in other areas (for example, the entire surface), the sacrificial layer may be left only in the area where the first electrode 131 and the second electrode 132 are to be formed and removed from other areas, thereby patterning the sacrificial layer to form the first sacrificial layer 121 and the second sacrificial layer 122. Furthermore, in this way, when sacrificial layers are formed in areas other than the areas where the first electrode 131 and the second electrode 132 are planned to be formed, the first sacrificial layer 121 and the second sacrificial layer 122 may not be immediately formed in the desired shape, but the sacrificial layers may be left in other areas, and when the conductive layer 13 is patterned as described below, the unnecessary sacrificial layers may be removed together with the unnecessary conductive layer 13, thereby forming the first sacrificial layer 121 and the second sacrificial layer 122 in the desired shape at this stage.

<Step (B)>
5(d), a first electrode 131 is formed on the surface of the first sacrificial layer 121 opposite to the substrate 11 side, and a second electrode 132 is formed on the surface of the second sacrificial layer 122 opposite to the substrate 11 side. At this time, in order to manufacture the electrode sheet 101, in addition to the first electrode 131 and the second electrode 132, a third conductive layer 133, a fourth conductive layer 134, a first axial conductive layer 135, and a second axial conductive layer 136 are also formed.

To achieve this, as shown in FIG. 5(b), a conductive layer 13 is formed on one surface 11a of the substrate 11 in the area where the first sacrificial layer 121 and the second sacrificial layer 122 are formed and in other areas. At this time, the conductive layer 13 may be formed on the entire surface of one surface 11a of the substrate 11, including the area where the first sacrificial layer 121 and the second sacrificial layer 122 are formed. The conductive layer 13 can be formed by a known method, such as a method of transferring a conductive layer 13 separately prepared in a location other than the substrate 11 to the desired area on one surface 11a of the substrate 11.

5C, a first contact pad 151 and a second contact pad 152 are separately formed in an area on the surface of the conductive layer 13 opposite to the substrate 11, where the first sacrificial layer 121 and the second sacrificial layer 122 are not formed. The first contact pad 151 and the second contact pad 152 can be formed by a known method such as a chemical vapor deposition (CVD) method.
By forming the first contact pads 151 and the second contact pads 152 at this stage, the electrode sheet 101 (electrode structure 1) can be manufactured efficiently.

Next, as shown in FIG. 5(d), the conductive layer 13 is patterned to form a first electrode 131 in which the first pore is not yet formed, a second electrode 132 in which the second pore is not yet formed, a third conductive layer 133, a fourth conductive layer 134, a first axial conductive layer 135, and a second axial conductive layer 136. At this stage, the first electrode 131, the second electrode 132, the third conductive layer 133, the fourth conductive layer 134, the first axial conductive layer 135, and the second axial conductive layer 136 may or may not have the final desired shape, and if they do not have the desired shape, they may be adjusted to the final desired shape in a later appropriate process.

Next, prior to step (C), as shown in FIG. 6(a), an insulating layer 14 is formed on one surface 11a of the substrate 11 in the areas where the first electrode 131, the second electrode 132, the third conductive layer 133, the fourth conductive layer 134, the first axial conductive layer 135, and the second axial conductive layer 136 are formed, as well as in other areas. At this time, the insulating layer 14 may be formed on the entire surface of one surface 11a of the substrate 11, including the areas where the first electrode 131, the second electrode 132, the third conductive layer 133, the fourth conductive layer 134, the first axial conductive layer 135, and the second axial conductive layer 136 are formed. The insulating layer 14 can be formed by a known method, such as a chemical vapor deposition (CVD) method.

Next, the photoresist layer is patterned on the insulating layer 14 by photolithography using a photoresist, by leaving the photoresist layer only in the formation region of the second electrode 132, the second axial conductive layer 136, the first axial conductive layer 135, the third conductive layer 133, and the fourth conductive layer 134, and removing the photoresist layer from the other regions. At this time, the photoresist layer is removed from the formation region of the second hole, which will be described later, among the formation region of the second electrode 132. Next, the second insulating layer 14 is formed on the entire surface of the side on which the photoresist layer is patterned on the substrate 11. The formation method of the insulating layer 14 at this time is the same as the formation method of the first insulating layer 14. Next, the photoresist layer is patterned on the second insulating layer 14 by photolithography using a photoresist, by leaving the photoresist layer only in the formation region of the first electrode 131, and removing the photoresist layer from the other regions. At this time, the photoresist layer is removed from the formation region of the first hole, which will be described later, among the formation region of the first electrode 131. Next, the insulating layer 14 that is not covered with the photoresist layer is removed. The insulating layer 14 can be removed by a known method, for example, etching with oxygen plasma. Next, the photoresist layer is removed. As a result, the first insulating layer 141, the second insulating layer 142, the third insulating layer 143, the fourth insulating layer 144, the first shaft portion 81, and the second shaft portion 82 are formed on one surface 11a of the substrate 11. At this time, the insulating layer 14 may be left between the first electrode 131 and the second electrode 132. At the stage shown in FIG. 5(d), if the first electrode 131, the second electrode 132, the third conductive layer 133, the fourth conductive layer 134, the first axial conductive layer 135, or the second axial conductive layer 136 does not have the final desired shape, these electrodes or conductive layers may be adjusted (patterned) to the desired shape at the same time as removing the insulating layer 14 by etching or the like.
As a result of the above, a first thin film 91 and a second thin film 92 are formed on one surface 11a of the substrate 11, as shown in FIG. 6B.

So far, we have described the formation of the first thin film 91, the second thin film 92, the first shaft portion 81, the second shaft portion 82, the third conductive layer 133, the fourth conductive layer 134, the third insulating layer 143, the fourth insulating layer 144, the first contact pad 151, and the second contact pad 152, but the formation method and formation order of these are not limited to those shown here.

6(c), a first support 161 is provided on the surface of the first shaft portion 81 opposite to the substrate 11 side, and a second support 162 is provided on the surface of the second shaft portion 82 opposite to the substrate 11 side. The first support 161 and the second support 162 can be provided on the first shaft portion 81 and the second shaft portion 82, respectively, by using a micromanipulator.
In this manner, the electrode sheet 101 is obtained.

If necessary, a protein layer as described above may be provided on either or both of the surface of the first insulating layer 141 in the first thin film 91 (the surface opposite the first electrode 131 side) and the surface of the second insulating layer 142 in the second thin film 92 (the surface opposite the second electrode 132 side).
The protein layer can be formed, for example, by contacting a protein solution such as an aqueous protein solution with an object on which the protein layer is to be formed, drying the solution, and physically adsorbing the protein to a desired location. More specifically, the protein solution can be brought into contact with the object on which the protein layer is to be formed by, for example, a method of immersing the object on which the protein layer is to be formed in the protein solution, a method of dripping the protein solution onto the object on which the protein layer is to be formed, a microcontact printing method, a patterning method using a microchannel, an inkjet printing method, or the like. In particular, among the five methods listed here, the methods other than the method of immersing the object on which the protein layer is to be formed in the protein solution are suitable for forming a protein layer only on either the surface of the first insulating layer 141 or the surface of the second insulating layer 142.
When a protein layer is provided on the surface of the first insulating layer 141 or the second insulating layer 142, a protein layer may or may not also be provided on the surface of the first pore 919 in the first insulating layer 141 or the surface of the second pore 929 in the second insulating layer 142.

Next, as shown in FIG. 7(a), the cells 7 to be cultured are seeded and held on the surface of the first insulating layer 141 in the first thin film 91. When the protein layer is provided on the surface of the first insulating layer 141, the cells 7 are seeded and held on the surface of the protein layer. The cells 7 on the surface of the first insulating layer 141 (first thin film 91) are prevented from entering the first pores 919 in the first thin film 91.

<Step (C)>
After the electrode sheet 101 is produced as described above, the first sacrificial layer 121 and the second sacrificial layer 122 are dissolved by applying a stimulus from the outside to the first sacrificial layer 121 and the second sacrificial layer 122, and the first electrode 131 formed on the first sacrificial layer 121 and the second electrode 132 formed on the second sacrificial layer 122 are liberated on the substrate 11. At the same time, the second electrode 132 is rolled into a cylindrical or semi-cylindrical shape, and the first electrode 131 is rolled into a cylindrical shape around the second electrode 132, forming a two-layer cylindrical structure 90 as shown in FIG. 7(b). This allows the desired electrode structure 1 to be obtained. The cell 7 is encapsulated in the two-layer cylindrical structure 90.

The method of applying the stimulus to the first sacrificial layer 121 and the second sacrificial layer 122 can be selected arbitrarily depending on the type of stimulus. For example, when applying the stimulus by applying a chemical substance, a solution such as an aqueous solution of the chemical substance may be brought into contact with the first sacrificial layer 121 and the second sacrificial layer 122. At this time, the electrode sheet 101 may be immersed in the solution. Since the solution does not affect the survival of the cells 7, the cells 7 are well encapsulated in the two-layered cylindrical structure 90.

When the first sacrificial layer 121 and the second sacrificial layer 122 are dissolved, the first thin film 91 is bent in a self-organizing manner and wound into a cylindrical shape due to the gradient of mechanical properties that occurs in the first electrode 131 and the first insulating layer 141 in the first thin film 91. Similarly, when the second sacrificial layer 122 is dissolved, the second thin film 92 is bent in a self-organizing manner and wound into a cylindrical shape due to the gradient of mechanical properties that occurs in the second electrode 132 and the second insulating layer 142 in the second thin film 92. Here, examples of the mechanical properties of the first electrode 131 and the first insulating layer 141 in the first thin film 91 include residual stress that occurs therein or elasticity in the thickness direction of these layers. Similarly, examples of the mechanical properties of the second electrode 132 and the second insulating layer 142 in the second thin film 92 include residual stress that occurs therein or elasticity in the thickness direction of these layers. At this time, the first thin film 91 rolls around the first shaft 81, and the second thin film 92 rolls around the second shaft 82. When the same bending stress is applied, the curvature of the first thin film 91 and the second thin film 92 is determined depending on their thickness. Therefore, for example, by making the thickness of the first insulating layer 141 and the thickness of the second insulating layer 142 different, the first thin film 91 and the second thin film 92 roll with different curvatures, forming a two-layer cylindrical structure 90.

The first thin film 91 rolls along the outer peripheral surfaces of the first support 161 and the second support 162, forming a cylindrical first thin film 910. The surface of the first thin film 910 contacts, and preferably adheres to, the outer peripheral surfaces of the first support 161 and the second support 162. On the other hand, the second thin film 92 rolls in the gap between the first support 161 and the second support 162, forming a cylindrical or semi-cylindrical second thin film 920.

So far, we have explained the manufacturing method for an electrode structure with a two-layer cylindrical structure, but electrode structures with a multi-layer cylindrical structure of three or more layers instead of two layers can also be manufactured in a similar manner.

The above describes an embodiment of the present invention in detail with reference to the drawings, but the specific configuration is not limited to this embodiment and includes designs that do not deviate from the gist of the present invention.

The present invention will be described in more detail below with reference to specific examples. However, the present invention is not limited to the examples shown below.

[Example 1]
<<Manufacture of electrode structure>>
A sodium alginate solution having a concentration of 1% wt/vol was applied by spin coating onto one side of a glass substrate (thickness 150 μm) made of borosilicate glass. The glass substrate after the application was immersed in a calcium chloride solution having a concentration of 0.1 mol/L to form a calcium alginate gel layer on the one side of the glass substrate. The glass substrate with the gel layer was taken out of the calcium chloride solution, washed with distilled water, and then naturally dried. Thus, a sacrificial layer was formed. Next, the gel layer (sacrificial layer) was left only in the areas on the glass substrate where the first and second electrodes were to be formed, and the gel layer was removed from the other areas, thereby patterning the gel layer on the glass substrate. Thus, a first sacrificial layer and a second sacrificial layer composed of the gel layer were formed on one side of the glass substrate (step (A)).

Single-layer graphene was grown on copper foil, the copper foil was etched, and the single-layer graphene was floated on distilled water. Then, using the glass substrate on which the first and second sacrificial layers had been formed, the single-layer graphene was scooped up from the surface of the glass substrate on which the first and second sacrificial layers had been formed, and the single-layer graphene was transferred onto the surface of the glass substrate on which the first and second sacrificial layers had been formed, so as to cover these sacrificial layers.

Next, two contact pads, i.e., a first contact pad and a second contact pad, were formed on the conductive layer by thermal evaporation through a mask, with a chromium layer and a gold layer stacked in that order from the glass substrate side. These contact pads were formed in areas near both ends of the conductive layer so as to sandwich the first electrode. Each of these contact pads had a rectangular planar shape and a size of 1 mm x 3 mm, with the chromium layer being 10 nm thick and the gold layer being 100 nm thick.

Then, the conductive layer is left integrally in the region where the first electrode is to be formed, the region where the third conductive layer is to be formed, and the region where the first axial conductive layer connecting them is to be formed, and further, the conductive layer is left integrally in the region where the second electrode is to be formed, the region where the fourth conductive layer is to be formed, and the region where the second axial conductive layer connecting them is to be formed, but these conductive layers left integrally are not in contact with each other. As a result, a first electrode without a first pore is formed on the surface of the glass substrate on which the first sacrificial layer and the second sacrificial layer are formed, so as to cover the first sacrificial layer, and a second electrode without a second pore is formed on the surface of the glass substrate on which the first sacrificial layer and the second sacrificial layer are formed, and further, the third conductive layer, the fourth conductive layer, the first axial conductive layer, and the second axial conductive layer are formed directly on the glass substrate (step (B)).

Next, an insulating layer (100 nm thick) made of chloroparaxylene was formed by depositing chloroparaxylene by chemical vapor deposition (CVD) on the entire surface of the side of the glass substrate where the first contact pad and the second contact pad were formed. Then, a photoresist was patterned on the insulating layer by photolithography using a photoresist, leaving the photoresist layer only in the formation areas of the second electrode, the second axial conductive layer, the first axial conductive layer, the third conductive layer, and the fourth conductive layer, and removing the photoresist layer from the other areas. At this time, the photoresist layer was removed from the formation area of the second hole in the formation area of the second electrode. Furthermore, a second insulating layer (150 nm thick) made of chloroparaxylene was formed by depositing chloroparaxylene by CVD on the entire surface of the side of the glass substrate where the photoresist layer was patterned. Then, the photoresist layer was patterned on the second insulating layer by photolithography using a photoresist, leaving the photoresist layer only in the region where the first electrode was formed and removing the photoresist layer from the other regions. At this time, the photoresist layer was removed from the region where the first hole was formed among the region where the first electrode was formed. Then, the insulating layer not covered with the photoresist layer was removed by etching with oxygen plasma, and a first hole penetrating the first electrode 131 and the first insulating layer 141 was formed in the laminate of the first electrode 131 and the first insulating layer 141, and a second hole penetrating the second electrode 132 and the second insulating layer 142 was formed in the laminate of the second electrode 132 and the second insulating layer 142, and the photoresist layer was removed by treatment with acetone. As a result, a first insulating layer (thickness 250 nm) was formed on the surface of the first electrode opposite the glass substrate side, a second insulating layer (thickness 100 nm) was formed on the surface of the second electrode opposite the glass substrate side, a first axial insulating layer (thickness 100 nm) was formed on the surface of the first axial conductive layer opposite the glass substrate side, a second axial insulating layer (thickness 100 nm) was formed on the surface of the second axial conductive layer opposite the glass substrate side, a third insulating layer (thickness 100 nm) was formed in the area where the first contact pad was not provided on the surface of the third conductive layer opposite the glass substrate side, and a fourth insulating layer (thickness 100 nm) was formed in the area where the second contact pad was not provided on the surface of the fourth conductive layer opposite the glass substrate side. Up to this point, the shapes of the first electrode, the second electrode, the first axial conductive layer, the second axial conductive layer, the third conductive layer, and the fourth conductive layer were adjusted to the final desired shape, and the first axis portion and the second axis portion were formed. 50 first pores with a diameter of 3 μm were formed, and 20 second pores with a diameter of 15 μm were formed. In this way, the first thin film and the second thin film were formed.

Next, with glass fibers placed as cylindrical supports on the first and second shaft portions, a cell suspension containing HUVECs was placed on the first thin film, thereby seeding HUVECs on the first thin film.
In this manner, an electrode sheet was obtained.

Next, the electrode sheet after seeding with the cells was contacted with an aqueous solution of alginate lyase, whereby the first sacrificial layer and the second sacrificial layer were dissolved, and the first electrode formed on the first sacrificial layer and the second electrode formed on the second sacrificial layer were liberated on the glass substrate, and the second electrode (second thin film) was rolled up in a semi-cylindrical shape, and the first electrode (first thin film) was rolled up in a cylindrical shape around the semi-cylindrical second electrode to form a two-layer cylindrical structure (step (C)).
In this manner, the intended electrode structure was produced, in which HUVECs were enclosed within a two-layer cylindrical structure.

<<Measurement of Potential Difference Between First Electrode and Second Electrode>>
In the cell-containing electrode structure obtained above, the potential difference between the first electrode and the second electrode was measured. The measurement result is shown in Figure 8. Furthermore, in the electrode sheet after seeding with cells, before forming the two-layer cylindrical structure, the potential difference between the first electrode and the second electrode was also measured in the same manner. The measurement result is shown in Figure 9.

As is clear from Figures 8 and 9, there was no significant difference in the measurement results of the potential difference before and after winding the first and second electrodes. In other words, no breaks or the like were observed in the first electrode, second electrode, first shaft portion, or second shaft portion during the manufacturing process of the electrode structure, and it was confirmed that the obtained electrode structure is capable of measuring the electrical resistance value of the cell sheet.

[Example 2]
<<Manufacture of electrode structure>>
An electrode sheet was produced in the same manner as in Example 1.
Next, a fibronectin layer was formed by coating the surface of the first thin film in the electrode sheet with fibronectin, and a cell suspension containing human umbilical vein endothelial cells (HUVECs) was brought into contact with the fibronectin layer on the first thin film to seed HUVECs on the fibronectin layer.
Next, the electrode sheet after seeding with HUVECs was contacted with an aqueous solution of alginate lyase, which dissolved the first and second sacrificial layers, liberated the first and second electrodes formed on the sacrificial layers onto the glass substrate, and rolled the second electrode (second thin film) into a semi-cylindrical shape, and rolled the first electrode (first thin film) into a cylindrical shape around the semi-cylindrical second electrode to form a two-layer cylindrical structure.
In this manner, the intended electrode structure was produced, in which HUVECs were enclosed within a two-layer cylindrical structure.

<<Creation of three-dimensional cultured vascular tissue>>
In the electrode structure obtained above, HUVECs were cultured to form a cell sheet between the first and second thin films in the two-layer cylindrical structure, and a three-dimensional cultured vascular tissue with a cylindrical structure was produced. Image data of the three-dimensional cultured vascular tissue obtained using a phase contrast microscope at this time is shown in Figure 10. At this point, a single-layered cylindrical structure had been formed by the cell sheet in the middle of extending.

The present invention can be used to create three-dimensional cultured tissues of cells.

1...electrode structure, 11...substrate, 11a...one side of substrate, 121...first sacrificial layer, 122...second sacrificial layer, 131...first electrode before being wound into a cylindrical shape, 132...second electrode before being wound into a semi-cylindrical shape, 1310...first electrode wound into a cylindrical shape, 1320...second electrode wound into a semi-cylindrical shape, 135...first axial conductive layer, 136...second axial conductive layer, 1410...first insulating layer wound into a cylindrical shape, 1420...second insulating layer wound into a semi-cylindrical shape, 81...first axial portion, 82...second axial portion, 90...two-layer cylindrical structure, 910...first thin film wound into a cylindrical shape, 920...second thin film wound into a semi-cylindrical shape

Claims (7)

A first electrode wound in a cylindrical shape; and a second electrode wound in a cylindrical or semi-cylindrical shape and provided in a non-contact state with the first electrode,
The first electrode surrounds the second electrode, and the first electrode and the second electrode form a multi-layer cylindrical structure;
An electrode structure, wherein a gap between the first electrode and the second electrode in the multilayer cylindrical structure serves as a cell culture area. a first insulating layer is laminated on a surface of the first electrode facing inward in a radial direction of the multilayer cylindrical structure to form a first thin film;
a second insulating layer is laminated on a surface of the second electrode facing inward in the radial direction of the multilayer cylindrical structure to form a second thin film;
The electrode structure according to claim 1 , wherein a gap between the first thin film and the second thin film serves as the cell culture portion. The first electrode is connected to a first shaft portion extending in a direction perpendicular to the winding direction of the first electrode,
3. The electrode structure according to claim 1, wherein the second electrode is connected to a second shaft portion extending in a direction perpendicular to the winding direction of the second electrode. the first shaft portion has a first shaft-shaped conductive layer, and the first electrode and the first shaft-shaped conductive layer are electrically connected;
The electrode structure according to claim 3 , wherein the second shaft portion has a second axial conductive layer, and the second electrode and the second axial conductive layer are electrically connected. The electrode structure according to claim 4, wherein the potential difference between the first electrode and the second electrode can be measured via the first shaft portion and the second shaft portion. The electrode structure according to any one of claims 2 to 5, wherein the first electrode, the second electrode, the first insulating layer, and the second insulating layer are optically transparent. A method for producing the electrode structure according to any one of claims 1 to 6, comprising the steps of:
A step (A) of forming a first sacrificial layer for laminating the first electrode and a second sacrificial layer for laminating the second electrode on one surface of a substrate that is non-conductive at room temperature;
A step (B) of forming the first electrode on a surface of the first sacrificial layer opposite to the substrate side, and forming the second electrode on a surface of the second sacrificial layer opposite to the substrate side;
and (C) applying a stimulus from outside the first sacrificial layer and the second sacrificial layer to dissolve the first sacrificial layer and the second sacrificial layer, thereby liberating the first electrode formed on the first sacrificial layer and the second electrode formed on the second sacrificial layer on the substrate, and winding the second electrode into a cylindrical or semi-cylindrical shape and winding the first electrode into a cylindrical shape around the second electrode, thereby forming the multilayer cylindrical structure.

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